Currently, the insecticidal gene widely used in the world for biological control of pests is Bt toxin gene of Bacillus thuringiensis (Bt) (such as: Cry1Ab, Cry1Ac, Cry1C and Cry1F et al.). Bacillus thuringiensis is insect pathogenic bacterium. The Bt toxin generated by it has a specific killing effect to many species of agricultural and forestry pests. Since Belgian Plant Genetic Systems first reported the success of transgenic Bt insect-resistant tobacco in 1987 till today, Bt gene has been transferred to main crops in the world, such as: maize, paddy, cotton, tomato, potato and tobacco. According to the statistics of International Service for the Acquisition of Agri-biotech Applications (ISAAA) in 2012, the area of transgenic Bt cotton grown in China has exceeded 3.9 million hectares, accounting for 71.5% of the total area of the cotton grown in China. However, following the application and generalization of transgenic Bt crops, its possible potential hazards in gene escape, change of microbial ecological structure of soil, drug resistance of species and harm to normal immune system have gradually aroused the attention of the society. “Diversity of Rhizospheric Microorganisms and Bacterial Physiological Groups of Transgenic Bt Maize” (Wang Min et al, Chinese Journal of Ecology, Issue 03 of 2010) and “Influence of Transgenic Bt Maize on Bacterial Quantity and Diversity of Soil” (Liu Ling et al, Journal of Ecology and Rural Environment, Issue 03 of 2011) analyzed the bacterial quantity and diversity of the soil in which transgenic Bt maize is grown indoors and outdoors respectively. The results all show significant difference between the transgenic Bt maize growing group and the blank control group.
“Cry1Ac protoxin from Bacillus thuringiensis sp. kurstaki HD73 binds to surface proteins in the mouse small intestine” (Vazquez-Padron et al., Biochem Biophys Res Commun, Issue 01, 2000) discovered that when intrinsic toxic protein of Bt and extrinsic toxic protein of Bt taken in by a mouse reached 10 mg/kg and 100 mg/kg, T cell ANAE positive rate, spleen index and macrophage phagocytosis of the mouse all were inhibited obviously during animal experiment. The more the intake is, the more obvious the inhibiting effect will be. This experiment also discovered that when the cumulative coefficient of Bt toxin protein in animal body was greater than 6.24, it might result in injury of liver, kidney and gastrointestinal tract and in liver and kidney, anomalies of cellular swelling and vacuolar degeneration could be observed and glomerular vascular epithelial lesion could be seen. Of course, it can't be excluded that they were caused by immunoreactions. Meanwhile, long-term use of Bt toxin protein at a large dose may also result in significant decrease of total white blood cells (WBC) and hemoglobin (HGB) of animals. This also indicates Bt toxin protein has obvious toxicity of immunosuppression. Therefore, developing substitute biological effectors with Bt toxin bioactivity (such as: Anti-idiotype antibody) is a research hotspot in biological pest development field.
As humanized antibody gene is derived from human, it has the advantage of gene homology with human immune system. It may avoid harm of its residue in food to human immune system after spray of its preparation or transgenic expression.
In 1974, Danish immunologist Jerne introduced the concept of Anti-idiotype antibody in his “Immune Network Theory”. Anti-idiotype antibody (hereinafter referred to as “Anti-Id”) refers to the specific antibody generated to address the idiotype (hereinafter referred to as “Id”) in the variable regions of antibody molecules. Bona, et al classified Anti-Id into four types (α, β, γ and δ) based on serological reaction between Id and Anti-Id as well as the function of AId. β-type Anti-Id has the effect of “internal image”, i.e.: has antigenic determinant same as (haptin) antigen, so it may have the function and bioactivity of antigen.
Currently, it is universally believed that Anti-Id with an effect similar to target antigen may be obtained by phage display technology through establishment of a phage antibody library, and specific screening. The process of screening specific antibody by phage display technology is called “Panning” and mainly includes four steps: binding, washing, eluting and amplification. Raats et al. adopted anti-cortisol monoclonal antibody coating as solid-phase antigen for direct screening. Before screening, a same species of negative monoclonal antibody is negatively screened to avoid screening recombinant antibody fragments bound to the constant region of antibody and successfully screen Anti-Id against cortisol. Goletz et al. also applied phage antibody display system and researched and compared the influence of different elution methods on Anti-Id fragment screening results. Of the eventually screened 96 clones, 28 were positive clones with Anti-Id characteristics. So far, no materials and products specific to substitutable Bt active effector, particularly Anti-Bt toxin type Anti-Id single-chain antibody (hereinafter referred to as “Anti-Id ScFvs”), have been reported.